Dual Functional Half-Sandwich Ru(II) Complexes: Lysosome-Targeting Probes and Anticancer Agents

Article abstract of DOI:10.1002/ejic.201801339

It is known that the proposed biologically active form of ruthenium is its oxidation state II other than oxidation state III, and ruthenium complexes offer the potential of a novel mechanism of action, reduced toxicity. Herein, three half-sandwich Ru

Full text of DOI:10.1002/ejic.201801339

A Journal of
Accepted Article
Title: Dual functional half-sandwich Ru(II) complexes: lysosome-
targeting probes and anticancer agents
Authors: Zhe Liu, JuanJuan Li, Deliang Kong, Meng Tian, Yao Zhao,
Zhishan Xu, Wenyuan Gao, and Yumin Zhou
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Eur. J. Inorg. Chem. 10.1002/ejic.201801339
Link to VoR: http://dx.doi.org/10.1002/ejic.201801339

European Journal of Inorganic Chemistry
10.1002/ejic.201801339
Dual functional half-sandwich Ru(II) complexes: lysosome-targeting probes and anticancer
agents
[
a]‡
[a]‡
[a]
[a]
[b]
[a,c]
Zhe Liu, * JuanJuan Li,
Deliang Kong,
Meng Tian,
Yao Zhao,
Zhishan Xu,
[a]
[a]
Wenyuan Gao, Yumin Zhou.
[
a]
Institute of Anticancer Agents Development and Theranostic Application, The Key Laboratory of Life-Organic
Analysis and Key Laboratory of Pharmaceutical Intermediates and Analysis of Natural Medicine, Department of
Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165, China.
[
b]
CAS Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy
of Sciences, Beijing 100190, PR China
[
c]
Department of Chemistry and Chemical Engineering, Shandong Normal University, Jinan 250014, China.
The first two authors are equal first authors.
‡
Abstract
It is known that the proposed biologically active form of ruthenium is its oxidation state II other
than oxidation state III, and Ruthenium complexes offer the potential of a novel mechanism of
II
6
action, reduced toxicity. Herein, three half-sandwich Ru complexes [(η -p-cym)Ru(N^N)Cl]PF
6
were designed and synthesized. Lysosomes are involved in various aspects of cancer cell
immortalization and cell death. Thus, lysosomes are attractive pharmacological targets for
selective killing of cancer cells. We demonstrated that Ru2 can accumulate in lysosomes. In
addition, A549 cell variability remained at 87.81% after exposure to Ru2 for 24 h at the working
concentration. Meanwhile, Ru2 exhibited relatively high photostability and suitability for
long-term tracking. At increased concentration after 24 h of exposure to the complex toward A549
cell line, Ru2 induced a high apoptotic rate, cell cycle arrest, mitochondrial membrane potential
loss, and reactive oxygen species overload. Ru2 integrated the anticancer properties and imaging
capabilities and is expected to be developed as a dual functional theranostic agent.
Keywords. Anticancer agents; Apoptosis; Lysosome-targeting probes; Photostability; Ruthenium.
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201801339
Introduction
Transition metal complexes remain a significant resource to produce chemical diversity in the
1-5
search for new diagnostic and therapeutic agents, for a relatively readily subtle or dramatic
change of the characteristics of the complex by alter-related ligands. Therefore, biological
applications of transition metal complexes have been greatly explored, especially in the anticancer
6-14
drug development.
Cisplatin (CDDP), oxaliplatin (OXA), and carboplatin represent the most
active and useful clinical transition metal anticancer agents, approximately nearly 50% of all
anticancer therapies globally. These drugs are especially effective in treating lung and ovarian
cancer. These successes demonstrated that utilization of metals in medicine may be a useful
strategy in drug design and development. However, similar to other cytotoxic drugs, problems of
platinum-based anticancer drugs, normal tissue toxicity, and drug resistance limit their future
1
5
use. These factors necessitate the exploration of new chemotype anticancer agents that have
1
6-20
metal and new mechanisms of action different from platinum.
Very recently, ruthenium (Ru) complexes have been reported to possess great potential in the
II
III
search for therapeutic agents. These agents not only showed rich redox chemistry (Ru and Ru )
2
1
but also had less toxicity than their platinum analogs. Recent study by Dyson has shown that
ruthenium arene complexes containing α-diimine ligands have appeared as potential anticancer
2
2
agents. Previous investigators have demonstrated that ruthenium compounds have displayed
2
3-25
potent anticancer activities,
KP1339) and have entered clinical trials.
e.g., NAMI-A) is one of the mitochondrion and cell surface target agents, indicating that the
mitochondrion, the cell surface, and other organelles targets have also been implicated in the
such as three ruthenium complexes (NAMI-A, KP1019, and
2
1, 26
In contrast to the nuclear targets, Ru anticancer drug
(
27-32
anticancer activity of Ru complexes.
In this regard, we are interested in the synthesis of novel
Ru (II) organometallic arene complexes based on N^N-chelating ligands. We also investigated the
effects of N^N-chelating ligands on the chemical and biological activities. N^N-chelating ligand
derivatives with different substitution patterns have recently drawn attention as promising catalyst
for polymerization. Incorporation of the electron withdrawing groups into the imino ligands can
3
3
also enhance both the catalytic activity and selectivity of the complex. In addition to promising
catalytic activity, N^N-chelating ligand derivatives provide the prospect for structural
diversification through metal complexation.
II
In this study, we choose the diimino as N^N-chelating ligands to construct half-sandwich Ru
6
complexes [(η -p-cym)Ru(N^N)Cl]PF , Ru1, Ru2, and Ru3, (Scheme 1) as theranostic agents.
6
We explored the cell toxicity, photobleaching and the mechanism of action (MOA) of these metal
complexes. The study MOA including catalytic hydride transfer analysis, cellular distribution and
II
uptake cell cycle, ROS, and apoptosis. The results suggested that three Ru complexes are
potential candidates for development as new therapeutic agents and long-term lysosome tracking
agents.
Results and Discussion.
Syntheses.
6
Dimeric μ-chloro-bridged complex [(η -p-cym)RuCl ] (dimer) formed upon conversion of
2
2
3
4
α-terpinene with RuCl under refluxing in absolute methanol. The N^N-chelating ligand were
3
synthesized by 2,3-butanedione and aniline derivative under the conditions of HOAc (1ml) and
MeOH refluxed for 12 h. Ru1, Ru2, and Ru3 were synthesized in high yields by stirring at room
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201801339
temperature the dinuclear ruthenium dimer and the corresponding ligands L -L in methanol under
1
3
N atmosphere and then purified by recrystallization (see Supporting Information and Scheme 2).
2
1
Ru1, Ru2, and Ru3 were identified by HNMR, elemental analysis and ESI-MS.
Scheme 1. Chemical Structures of ruthenium(II) complexes
^
Scheme 2. Synthesis ruthenium (II) arene complexes containing N N ligands.
Photo Physical Properties and Hydrolysis Studies.
At the beginning, we tested the UV–Vis spectra of Ru1, Ru2, and Ru3 acquired in
phosphate-buffered solutions at 298 K (Figure S1). The intense absorption bands of these
complexes range at approximately at 250–600 nm. These bands could be assigned to the mixed
1
charge-transfer modes, such as singlet and triplex metal-to-ligand charge transfer ( MLCT and
3
28, 35
MLCT), mixed ligand-centered transition, and ligand-to-ligand charge transfer.
hydrolysis of complexes Ru1, Ru2, and Ru3 in 10% MeOH/90% H O (v/v) by using UV–Vis at
We tested the
2
298 K (Figure S2 in the Supporting information). No clear change was observed over 6 h for
those complexes, suggesting that Ru1, Ru2, and Ru3 do not hydrolyze at this condition.
Cytotoxicity.
The in vitro cytotoxicity of the organometallic complexes and cisplatin (control) was screened in
vitro against HeLa (human cervical cell cancer) and A549 (nonsmall cell lung cancer) cell lines by
using MTT assay after 24 h or 72 h treatment. The half-maximal inhibitory concentration (IC )
5
0
values are listed in Table 1 and shown in Figure S3 in the Supporting Information, wherein
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201801339
cisplatin was used as the standard control. Ru1, Ru2, and Ru3 showed medium anticancer activity
against HeLa cell line after 24 h incubation. However, the recorded IC values of the complexes
5
0
showed comparable activity toward HeLa cell line after 72 h incubation. These complexes
exhibited better anticancer activity against A549 cell line compared with HeLa cell line whether at
24 h or 72 h. For complex Ru2 (22.2 µM ) equipotent to cisplatin (21.3 µM ) after 24 h, Ru1 and
Ru3 were of intermediate value against A549 cell line. However, all complexes showed good
activity when incubated for 72 h for A549 cell lines. Notably, Ru2 demonstrated 4.9-fold greater
potency after 72 h incubation than that after 24 h incubation in A549 cells and approximately
1.6-fold greater potency than cisplatin. Previous cytotoxicity studies on ruthenium(II) p-cymene
anticancer complexes show that a marked increase in the cytotoxicity when aspirin and valproic
2
2
acid was introduced on α-diimine ligands. The resulting IC values showed that substitution of
5
0
the alterations to the chelating ligand had minimal effect on inhibitory activity in this study.
Next, the antiproliferative activities of Ru2 against BEAS-2B (human bronchial epithelial normal
cell) were also evaluated by MTT assay after 72 h incubation, however, no selectivity was
observed for the complex between cancer cells versus BEAS-2B normal cell (IC = 8.0 ± 0.5 μM
5
0
towards BEAS-2B normal cell).
Table 1. Cytotoxicities (IC ) of Ru1-Ru3 and Cisplatin towards different cell lines.
50
IC (µM)
5
0
Complex
BEAS-2B
2 h
A549
24 h
HeLa
7
72 h
24 h
72 h
Ru1
Ru2
27.7±1.0 5.9±0.4
22.2±0.9 4.5±0.7
34.5±0.2 7.8±0.5
21.3±1.7 7.2±1.0
33.6±0.8
28.9±1.7
37.8±1.0
7.5±0.2
8.5±0.2
5.8±0.5
9.7±0.3
5.5±0.4
8.0±0.5
Ru3
Cisplatin
a
Cells were treated with different concentrations of the ruthenium complexes and cisplatin for 24
h or 72 h. Cell viability was determined by the MTT assay, and data were calculated as described
in the experimental section. Data are presented as means ± SD obtained in at least three
independent experiments.
Partition Coefficients (log P).
The in vitro antiproliferative activity and cellular uptake levels of metal complexes were
consistent with their relative lipophilicity. The log P values determined for Ru1–Ru3 were from
0.7 to 3.3 (Table 2). To test the log P of the chloride complexes, NaCl (50 mM) was used for
hydrolysis inhibition. The anticancer potential and hydrophobicity of the complexes were
correlated in this study. Ru2 displayed higher hydrophobicity and the most antiproliferative
activity than Ru1 and Ru3. Similarly, the same trend that the cytotoxicity of the respective
2
2
complexes tend to correlate with the observed lipophilicity that they provide reported by Dyson.
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201801339
a
Table 2. Log P Values for Ru1-Ru3
log P
Complex
mean
0.36
1.41
0.32
SD
Ru1
Ru2
Ru3
0.01
0.19
0.09
a
Results are the means of three independent experiments and areexpressed as means ±SDs.
Catalytic oxidation of NADH.
+
The coupled nicotinamide adenine dinucleotide (NADH) and its oxidized form NAD play
crucial roles as the cofactors in a wide range of biological and biocatalyzed processes, such as
36
III
energy metabolism and cell death. Sadler and co-workers reported that the aqua Ir
cyclopentadienyl complexes can accept a hydride from the coenzyme NADH to catalytically
+
convert NADH to NAD and can be linked to ROS production that can induce severe or sustained
3
7, 38
ROS stress and directly induces cell death.
Therefore, reactions between metal complexes
Ru1, Ru2, and Ru3 and NADH were investigated. NADH (100 µM) in a solution of 10%
MeOH/90% H O (v/v) was used as the control to evaluate the catalytic activity of the complexes.
2
Moreover, the catalytic ability of complexes Ru1, Ru2, and Ru3 with NADH was monitored
under the same test conditions by UV–Vis at 298 K (Figure 1, Figure S4). The detection principle
+
was based on the UV absorption difference at 339 nm when the conversion of NADH to NAD the
absorption at 339 nm decreased. The turnover numbers (TONs) of Ru1, Ru2, and Ru3 were
calculated, and the results showed that Ru1 and Ru2 had similar TONs (Ru1:7.1, Ru2:7.4) and
approximately 2-fold larger than Ru3.
Figure 1. (A) UV-Vis spectra of the reaction of NADH (100 µM) with Ru2 (1 µM) in 10%
MeOH/90 % H O (V:V) at 298 K for 8 h; (B) The TONs of as-synthesized complexes. The arrows
2
show changes in absorbance spectra over time.
Cellular Uptake.
Through the above experiments, Ru2 has high inhibitory activity, hydrophobicity, and catalytic
activity. Thus, Ru2 was selected to carry out further investigations to describe their mechanism of
action. Then, we studied the cellular uptake mechanisms of Ru2. The cellular uptake mechanisms
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201801339
include energy-dependent or energy-independent pathways. Energy-dependent pathways can also
be divided into active transport and endocytosis. Active transport and endocytosis can be mediated
by metabolic inhibitor CCCP and chloroquine (an endocytosis modulator) respectively. Confocal
microscopy observations indicated that Ru2 incubation with A549 cells upon treatment with
CCCP or at 4 °C can lead to reduced cellular uptake efficiency (Figure 2). However, chloroquine
showed no effect on the Ru2 ability to cross the plasma membrane. The results indicated that Ru2
entering A549 cells do not rely on the endocytic pathways but possibly through an
energy-dependent pathway, e.g., via active transport.
Figure 2. Cellular uptake mechanisms of Ru2. Confocal images of A549 cells after incubation
with Ru2 (10 μM) under different conditions. (a) Cells were incubated with Ru2 (10 μM) at 37 °C
for 1h. This served as a control. (b) Cells were incubated with Ru2 (10 μM) at 4 °C for 1h. (c)
Cells were preincubated with metabolic inhibitors CCCP (50 uM) for 1 h at 37 °C and then
incubated with Ru2 (10 μM) at 37 °C for 1h. (d) Cells were preincubated with chloroquine (50
μM) for 1 h at 37 °C and then incubated with Ru2 (10 μM) at 37 °C for 1h. Emission was
collected at 550 ± 20 nm upon excitation at 488 nm. Scale bars: 10 μm
Cellular Localization.
The specific cellular target of the complexes determines the initial interactions between cells and
complexes and can be observed by confocal microscopy. Considering that lysosomes are involved
in various aspects of cancer cell immortalization and cell death, they are emerging as attractive
3
9, 40
pharmacological targets for selective killing of cancer cells.
The subcellular localization of the
complexes was confirmed by confocal microscopy and an inductively coupled plasma mass
spectrometry (ICP-MS) assay (Figure 3). We probed the specificity of Ru2 localization with
commercial mitochondria staining dye MitoTracker Deep Red (MTDR) and lysosomes staining
dye Lyso Tracker Deep Red (LTDR). As shown in Figures 3A and B, the LTDR signal correlated
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201801339
well with Ru2, providing high Pearson’s colocalization coefficient (PCC) at different times (1 h:
PCC=0.88, 3 h: PCC=0.90, 8 h: PCC=0.86, and 15h: PCC=0.84). However, minimal overlap is
observed for Ru2 (1 h: PCC=0.06, 3 h: PCC=0.04, and 15 h: PCC=0.07) with MTDR. In the
ICP-MS assay, a majority of the Ru2 localized in the cell membrane after 15 h of co-incubation
(Figure 3C). In general, lysosomes are a semimembrane vesicular structure of organelles. The
results were consistent to the observations of confocal microscopy images. Both of the assays
confirmed that Ru2 can specifically accumulate in the lysosomes for a long-term.
Figure 3. Determination of intercellular localization of Ru2 by laser confocal microscope. (A)
Selective imaging of A549 cells incubated with Ru2 (10 μM) for 1 h, 5 h and 8 h; then incubated
with MTDR (MitoTracker Deep Red) (75 nM) for 30 min at 37°C. The green and red fluorescence
represent Ru2 and mitochondria, respectively. (B) Selective imaging of A549 cells incubated with
Ru2 (10 μM) for 1 h, 5 h, 8 h and 15 h; then incubated with LTDR (Lyso Tracker Deep Red) (100
nM) for 30 min at 37°C. The green and red fluorescence represent Ru2 and the lysosome,
respectively. Ru2: λ =488 nm; λ = 550 ± 20 nm. MTDR: λ = 644 nm; λ = 660 ± 20 nm.
ex
em
ex
em
LTDR: λ = 594 nm; λ = 650 ± 20 nm. Scale bar: 10 μm. (C) Ruthenium content of the Nuclear
ex
em
Chromatin, Nucleus, Cytoskeleton, Cytosol and Membrane fractions of A549 cells after 24 h of
exposure to 10 μM Ru2. Results are the means of two independent experiments in triplicate and
are expressed as means ± SDs.
Apoptosis Assay and Cell Cycle Analysis.
Apoptosis is a common cell death pathway and reported to be the primary pathway for cell
2
death. To determine the relationship between cytotoxic activity and apoptosis, we treated the lung
cancer cells with Ru2 at 1, 2, and 3 equipotent concentrations of IC for 24 h and tested by flow
5
0
cytometry (Figure 4 and Table S1 in the Supporting Information). As shown in Figure 4, only a
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201801339
small portion of complex-treated cells (10.89%) are in late apoptosis at 1 x IC50 compared with
control (5.22%), and as increased complex concentration the percentage of cells in the apoptotic
phase dramatically added. A high apoptotic rate (75.76%) at late apoptotic phase can be achieved
for 3 × IC of Ru2 after 24 h co-incubation
50
.
To investigate whether the inhibition of cancer cell proliferation is associated with cell cycle
arrest, the cell cycle arrest in A549 cells exposed to Ru2 was observed using flow cytometry
(Figure 4, Table S2 in the Supporting Information). The cell cycle progression was analyzed at
0.25, 0.5, and 1 equipotent concentrations of IC50 of Ru2 for 24 h. The results showed that
compared with vehicle treatment, upon exposure of the cells to Ru2, a dose-dependent increase in
the proportion of G /G phase was observed (from 59.63% to 80.02%). The percentages of cells in
0
1
the G /G phase of the cell cycle increased 20.39%. This result demonstrated the complex
0
1
disturbance of the cell cycle at the G /G phase.
0
1
Figure 4. (A) Apoptotic population in A549 cells treated with Ru2 (1, 2, and 3 × IC ) for 24 h.
5
0
FACS analysis using AnnexinV-FITC/PI. The experiment is repeated three times and
representative photographs are displayed. And Columns, mean values of three independent
experiments. Data are presented as mean ± S.D. (B) Effect of Ru2 (0.25, 0.5, and 1 × IC ) on cell
5
0
cycle of A549 cells by FACS analysis exposed for 24 h. left: FACS analysis. A representative
experiment of these three independent experiments is demonstrated. Right: percentage of A549
cells in every phase of cell cycle upon different concentration treatment. The data are expressed as
the mean ± SD from three independent experiments.
Mitochondrial Membrane Potential (Δψ ) Changes and Induction of Intracellular Reactive
m
Oxygen Species (ROS).
The mitochondrion is an essential component of the intrinsic apoptotic signaling pathway. Thus,
we further investigated whether complexes induce apoptosis through the mitochondrial damage by
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201801339
41
measuring the MMP changes. The MMP loss and the ROS overload are the main pathways in
1
0, 42
the mitochondria to carry out cell death.
We investigated the intracellular MMP loss and ROS
production in Ru2-treated A549 cells by using fluorescent probe JC-1 and 2,7-dichlorofluorescein
diacetate (DCF-DA) by flow cytometry respectively. Fluorescence change from red to green
indicated a decrease in MMP. Ru2 (at concentrations of 0.25, 0.5, 1, and 2 × IC for 24 h) affects
5
0
the lost MMP in a dose-dependent manner, as shown in Figure 5A. In addition, the percentage of
cells with mitochondrial membrane depolarization increased from 6.91% to 73.81% after 24 h
treatment. The cause of ROS production was tested using DCF-DA, as shown in Figure 5B. A
significant increase in a dose-dependent manner was also observed. This finding was consistent
with the lost MMP results.
Figure 5. (A) The effect of Ru2 (0.25, 0.5, 1, and 2 × IC ) for 24 h on mitochondrial membrane
5
0
potentials were determined with JC-1 staining by flow cytometry. left: FACS analysis. One
representative profile out of three replicates. Right: The bar graph shows the percentage of JC-1
red fluorescence and JC-1 green fluorescence in A549 cells. Each point represents the mean ± SD
of three replicates. (B) Left: Effects of Ru2 (0.25 and 0.5 × IC ) for 24 h on ROS levels in A549
5
0
cells by flow cytometry. A representative experiment of these three independent experiments was
demonstrated. Right: The bar graph shows the mean fluorescence intensity of the generated ROS
in A549 cells. Data are presented as mean ± S.D.
Photobleaching.
The cell variability of Ru2 at 1 × IC (the working concentration) remains to be 87.81% after
5
0
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201801339
being exposed to A549 cells for 24 h (Table S1) and can specifically target lysosomes for a long
time (Figure 3B). Thus, we explored its potential as a dual-functional theranostic agent. In general,
a major shortcoming of a large amount of fluorescent organic bioprobes is its sensitivity to
photobleaching. As shown in Figure 6, the signal loss percentages after 20 scans were measured
to be 56.4% and 75.8% for Ru2 and LysoTracker Green, respectively. The results indicated that
Ru2 has relatively high cell variability and photostability. This condition is suitable for long-term
tracking.
Figure 6. Comparison of photostability between Ru2 (22.2 µM ) and LysoTracker Green (100 nM).
(A) Confocal fluorescence images of live A549 cells stained with Ru2 (λex = 488 nm) or
LysoTracker Green (λ = 488 nm) under serial excitation. (B) Luminescence decay curves of Ru2
ex
and LysoTracker Green after the same serial scans. The signals of Ru2 and MitoTracker Green
were collected at 520 ±20 nm.
Conclusion
II
6
In summary, three half-sandwich Ru complexes [(η -p-cym)Ru(N^N)Cl]PF , namely, Ru1, Ru2,
6
and Ru3 as theranostic and specifically lysosomes target agents, were synthesized. Ru2 is the
most potential complex that has high inhibitory activity, higher hydrophobicity and catalytic
activity compared with the other two complexes, along with a high apoptotic rate (75.76%), cell
cycle arrest, the loss of MMP and the overload of ROS can be achieved for Ru2 at higher
concentration after 24 h However, cell variability of Ru2 at 1× IC (22.2 μM, the working
.
50
concentration) remains to be 87.81% after exposed to A549 cells for 24 h and is suitable for
long-term tracking of specific target lysosomes. Meanwhile, Ru2 had relatively high
II
photostability. The results suggested that these Ru complexes are potential candidates for
development as a dual-functional theranostic agent.
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201801339
Experimental section
Materials and Instrumentation. Unless otherwise noted, all manipulations were performed using
standard Schlenk tube techniques under nitrogen atmosphere. hydrated RuCl ·nH O (≥99%
3
2
purity), octan-1-ol (≥99%), and Nitric acid (72%), glyoxal, 40 wt.% solution in H O, p-toluidine,
2
p-anisidine, 4-fluoroaniline, 4-chloroaniline, 4-methoxyaniline, were purchased from
Sigma-Aldrich. Dimer and ligand were prepared as described. For the biological experiments,
BSA, carbonyl cyanide m-chlorophenylhydrazone (CCCP), DMEM medium, fetal bovine serum,
penicillin/streptomycin mixture, trypsin/EDTA, and phosphate-buffered saline (PBS) were
purchased from Sangon Biotech. MTDR (Life Technologies), LTDR (Life Technologies), MTT
(
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich), Annexin V-FITC
Apoptosis Detection Kit (Sigma-Aldrich), JC-1 (Sigma-Aldrich), PBS (Sangon Biotech), PI
Sigma-Aldrich) are all used as received. Testing compounds was dissolved in DMSO and diluted
(
with the tissue culture medium before use.
Syntheses.
Synthesis of the ligands (L -L ).
1
3
Ligands L -L was synthesized according to the literature.
1
3
For synthesis of ligand L , a mixture solution of 2,3-butanedione (0.0115 mol),
1
4-methoxyaniline (0.0235 mol), HOAc (1ml) and MeOH (20 ml) was refluxed for 12 h, after
which the resulting suspension was filtered and the solid product washed with MeOH, until the
1
washing phase remained bright yellow and dried in vacuum. Yield: 2.23g 65.5%. H NMR (500
MHz, CDCl ) δ6.91 (d, 4H, J = 7.6 Hz), 6.78 (d, 4H), 3.84 (s, 6H), 2.17 (s, 6H). Anal. Calcd for
3
C H N O : C, 72.95; H, 6.80; N, 9.45; Found: C 72.90, H 6.85, N 9.42.
1
8
20
2
2
For synthesis of ligand L , a process similar to that of L was adopted. 2,3-butanedione
2
1
1
(
0.023 mol), 4-chloroaniline (0.046 mol) Yield: 6.00g 85.6%. H NMR (500 MHz, CDCl ) δ 7.175
3
(
d, 4H, J = 7.6 Hz), 6.70 (d, 4H, J = 8.0 Hz), 2.347 (s, 6H). Anal. Calcd for C H Cl N : C, 62.97;
1
6
14
2
2
H, 4.62; N, 9.18; Found: C 62.95, H 4.65, N 9.23.
For synthesis of ligand L , a process similar to that of L was adopted. 2,3-butanedione (0.023
3
1
1
mol), 4-fluoroaniline (0.046 mol) Yield: 4.75g 75.9%. H NMR (500 MHz, CDCl ) δ 7.175 (d, 4H,
3
J = 7.6 Hz), 6.70 (d, 4H, J = 8.0 Hz), 2.148 (s, 6H). Anal. Calcd for C H F N : C, 70.58; H, 5.18;
1
6
14
2
2
N, 10.29; Found: C 70.60, H 5.15, N 10.26.
6
Synthesis of the [(η -p-cymene)Ru(N^N)Cl]PF .
6
6
General method: A solution of dimer [(η -p-cymene)RuCl ] (0.05 mmol) and the
2
2
corresponding diimine base ligands L -L (0.10mmol) in methanol (20 ml) in a Schlenk bottle was
1
3
Stirring at room temperature in an N atmosphere for 10 h, cool to room temperature. NH PF
6
2
4
(
0.42 mmol) was added after constant stirring 5h. Methanol was removed under reduced pressure,
1
filter and recrystallized from dichloromethaneto/diethyl ether. The H NMR (500.13 MHz, CDCl3
or DMSO-d ) peak integrals of complexes 1, 2 and 3 are shown as follows.
6
6
1
[
(η -p-cymene)Ru(L )Cl]PF (1) Yield: 17.3 mg, 48.6%. H NMR (500 MHz, DMSO) δ 7.40 (s,
1 6 .
4
H), 7.18 (d, J = 9.1 Hz, 4H), 5.14 (d, J = 6.5 Hz, 2H), 5.09 (d, J = 6.4 Hz, 2H), 3.87 (s, 6H), 2.46
1
3
–
2.44 (m, 1H), 2.31 (s, 6H), 2.03 (s, 3H), 1.02 (d, J = 6.9 Hz, 6H). C NMR (126 MHz, DMSO)
δ 177.07 (s), 159.39 (s), 144.54 (s), 88.73 (s), 87.52 (s), 56.02 (s), 30.89 (s), 22.04 (s), 20.60 (s),
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201801339
6
1
8.66 (s). Anal. Calcd for (η -p-cymene)Ru(L )Cl] PF (712.10): C, 47.23; H, 4.81; N, 3.93.Found:
1 6
6
+
C, 47.25; H, 4.86; N, 3.89. MS: m/z 567.14 [(η -p-cymene)Ru(L )Cl] .
1
6
1
[
(η -p-cymene)Ru(L )Cl]PF (2) Yield: 14.2. mg, 39.2%. H NMR (500 MHz, DMSO) δ 7.73 (d,
2 6 .
J = 8.8 Hz, 4H), 7.46 (s, 4H), 5.20 (dd, J = 22.1, 6.4 Hz, 4H), 2.32 (s, 6H), 2.03 (s, 3H), 1.03 (d, J
13
=
6.9 Hz, 6H). C NMR (126 MHz, DMSO) δ 178.08 (s), 149.86 (s), 133.40 (s), 110.14 (s),
1
03.14 (s), 88.75 (s), 87.13 (s), 30.99 (s), 22.02 (s), 20.76 (s), 18.64 (s). Anal. Calcd for
6
[(η -p-cymene)Ru(L )Cl]PF (722.00): C, 43.32; H, 3.91; N, 3.89. Found:C, 43.36; H, 3.93; N,
1
6
6
+
3.83. MS: m/z 575.04 [(η -p-cymene)Ru(L )Cl] .
2
6
1
[
(η -p-cymene)Ru(L )Cl]PF (3) Yield: 14.7. mg, 42.8%. H NMR (500 MHz, DMSO) δ 7.51 (t,
3
6
.
J = 7.4 Hz, 8H), 5.19 (dd, J = 17.4, 6.5 Hz, 4H), 2.32 (s, 6H), 2.02 (s, 3H), 1.02 (d, J = 6.9 Hz, 6H).
1
3
C NMR (126 MHz, DMSO) δ 177.99 (s), 160.82 (s), 147.56 (s), 110.03 (s), 102.69 (s), 88.92 (s),
6
8
7.13 (s), 30.92 (s), 21.97 (s), 20.71 (s), 18.56 (s). Anal. Calcd for [(η -p-cymene)Ru(L )Cl]PF
3
6
(
688.06): C, 45.39; H, 4.10; N, 4.07. Found:C, 45.36; H, 4.13; N, 4.10. MS: m/z 543.10
6
+
[(η -p-cymene)Ru(L )Cl] .
3
1
NMR Spectroscopy. H NMR spectra were acquired in 5 mm NMR tubes at 298 K on Bruker
1
1
DPX 500 ( H = 500.13 MHz) spectrometers. H NMR chemical shifts were internally referenced
to (CHD )(CD )SO (2.50 ppm) for DMSO-d , CDCl (7.26 ppm) (for chloroform-d ). All data
2
3
6
3
1
processing was carried out using XWIN-NMR version 3.6 (Bruker UK Ltd.).
UV-Vis Spectroscopy. A TU-1901 UV-Vis recording spectrophotometer was used with 1
cm path-length quartz cuvettes (3 mL). Spectra were processed using UVWinlab software.
Experiments were carried out at 298 K unless otherwise stated.
Measurement of Lipophilicity LogP . log P was calculated as the logarithmic ratio of
o/w
o/w
metal concentration in n-octanol to that in aqueous phase. Octanol-saturated water (OSW) and
water-saturated octanol (WSO) were prepared using analytical grade octanol and 50 mM aqueous
NaCl solution (to suppress hydrolysis of the chloride complexes). Aliquots of stock solutions of
metal complexes in OSW were added to equal volumes of WSO and shaken in an IKA Vibrax
VXC basic shaker for 4 h at 500 g/min to allow partition at ambient temperature (∼298 K). The
aqueous layer was carefully separated from the octanol layer for metal complex analysis. Metal
was quantified from aliquots taken from the octanol-saturated aqueous samples before and after
partition. Partition coefficients of metal complexes were calculated using the equation log P = log
([M]WSO /[M]OSW ), where [M]WSO was obtained by subtraction of the metal content of the aqueous
layer after partition from the metal content of the aqueous layer before partition.
Reaction with NADH. The reaction of complex (ca. 1 µM ) with NADH (100 µM ) in 50%
MeOH/50% H O (v/v) was monitored by UV-Vis at 298 K after various time intervals. TON was
2
calculated from the difference in NADH concentration after 9 h divided by the concentration of
metal complex catalyst. The concentration of NADH was obtained using the extinction coefficient
-1
-1
ε339 = 6220 M cm .
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201801339
Cell Culture. A549 cervical carcinoma cells were obtained from Shanghai Institute of
Biochemistry and Cell Biology (SIBCB) and were grown in Dubelco's Modified Eagle Medium
DMEM). All media were supplemented with 10% fetal bovine serum, and 1%
penicillin-streptomycin solution. All cells were grown at 310 K in a humidified incubator under a
(
5
% CO atmosphere.
2
Viability assay (MTT assay). After plating 5000 A549 cells per well in 96-well plates, the
cells were preincubated in drug-free media at 310 K for 24 h or 72 h before adding different
concentrations of the compounds to be tested. In order to prepare the stock solution of the drug,
the solid complex was dissolved in DMSO. This stock was further diluted using cell culture
medium until working concentrations were achieved. The drug exposure period was 24 h or 72 h.
-1
Subsequently, 15 µL of 5 mg mL MTT solution was added to form a purple formazan.
Afterwards, 100 μL of dimethyl sulfoxide (DMSO) was transferred into each well to dissolve the
purple formazan, and results were measured using a microplate reader (DNM-9606, Perlong
Medical, Beijing, China) at an absorbance of 570 nm. Each well was triplicated and each
experiment repeated at least three times. IC values quoted are mean ± SEM.
5
0
Localization Experiments. Localization of complex (10 μM) with mitochondria or lysosome
were examined by means of MTDR (MitoTracker Deep Red) (Molecular Probes), a
mitochondria-specific dye and LTDR (LysoTracker Deep Red), a lysosome-specific dye. Briefly,
A549 cells were seeded into 6-well plates (Greiner, Germany) for confocal microscopy. After
cultured overnight, a 1 mM complex stock solution made in DMSO was diluted to 10 μM working
concentration in cell medium (DMEM, 5% FCS). Staining of mitochondria/lysosome was
accomplished by adding a 75 nM/100 nM final concentration of MTDR/LTDR to the culture
medium for the last 30 min of complex incubation. The medium was removed and washed three
times with ice-cold PBS, and then viewed immediately under a confocal microscope (Zeiss
LSM880 NLO). The excitation and emission bands of each compound were selected as follows
for complex: λ =488 nm; λ = 550 ± 20 nm. MTDR: λ = 644 nm; λ = 660 ± 20 nm. LTDR:
ex
em
ex
em
λex = 594 nm; λem = 650 ± 20 nm.
Inductively coupled plasma mass spectrometry (ICP-MS). A549 cells were seeded in 90 mm
dishes for 24 h (three dishes were prepared per compound tested). The media was removed and
replaced with fresh media containing the tested complex (5 μM) for 24 h. After the removal of the
culture media and rinse with 1 mL of PBS buffer (1X), the cells were treated with 500 μl of 0.25%
trypsin and centrifuged at 1000 rpm. The cells were counted, one half of the cells were centrifuged,
quickly washed with PBS, and stored at 253 K for determination of total cell accumulation of
ruthenium. Another half of the samples was used for cytosol and nucleus, using a nuclear and
cytoplasmic protein extraction kit (Shanghai Sangon Biological Engineering Technology &
Services Co. Ltd.). The samples were nitrolysis with concentrated HNO at 95 ̊C for 2 h, H O at
2 2
3
95 ̊C for 1.5 h and concentrated HCl at 37 C̊ for 0.5 h. Finally, the solution was diluted to 2 mL
with MQ water and the Pt content was measured by the inductively coupled plasma mass
spectrometer (ICP-MS; VG Elemental).
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201801339
Cellular Uptake. A549 cells were seeded in 6-well plates for 24 h and preincubated with CCCP
50 μM) or chloroquine (50 μM) for 1 h. The medium was removed and the cells were then
(
incubated with complex (10 μM) for 1 h. To investigate the impact of temperature on cellular
uptake, the cells were incubated at 4 °C or 37 °C for 1 h. In each case, the cells were washed three
times with ice-cold PBS and visualize by confocal microscopy (Zeiss LSM880NLO) immediately.
λ = 488 nm, λ = 550 ± 20 nm.
ex
em
Induction of Apoptosis. Flow cytometry analysis of apoptotic populations of A549 cells
caused by exposure to metal complexes was carried out using the Annexin V-FITC Apoptosis
Detection Kit (Beyotime Institute of Biotechnology, China) according to the supplier’s
6
instructions. Briefly, A549 cells (1.5 ×10 /2 mL per well) were seeded in a six-well plate. Cells
were preincubated in drug-free media at 310 K for 24 h, after which drugs were added at
concentrations of 1 × IC50, 2 × IC and 3 × IC . After 24 h of drug exposure, cells were collected,
50
50
washed once with PBS, and resuspended in 195 μL of annexin V-FITC binding buffer which was
then added to 5 μL of annexin V-FITC and 10 μL of PI, and then incubated at room temperature in
the dark for 15 min. Subsequently, the buffer placed in an ice bath in the dark. The samples were
analyzed by a flow cytometer (ACEA NovoCyte, Hangzhou, China).
6
Cell Cycle Analysis. A549 cells at 1.5 × 10 per well were seeded in a six-well plate. Cells
were preincubated in drug-free media at 310 K for 24 h, after which drugs were added at
concentrations of 0.25 × IC50, 0.5 × IC and IC . After 24 h of drug exposure, supernatants were
50
50
removed by suction and cells were washed with PBS. Finally, cells were harvested using
trypsin-EDTA and fixed for 24 h using cold 70 % ethanol. DNA staining was achieved by
resuspending the cell pellets in PBS containing propidium iodide (PI) and RNAse. Cell pellets
were washed and resuspended in PBS before being analyzed in a flow cytometer (ACEA
NovoCyte, Hangzhou, China) using excitation of DNA-bound PI at 488 nm, with emission at 585
nm. Data were processed using NovoExpress™ software. The cell cycle distribution is shown as
the percentage of cells containing G /G , S G /M and Sub-G phase DNA as identified by
0
1
2
1
propidium iodide staining.
ROS Determination. Flow cytometry analysis of ROS generation in A549 cells caused by
exposure to metal complexes was carried out using the Reactive Oxygen Species Assay Kit
(
Beyotime Institute of Biotechnology, Shanghai, China) according to the supplier’s instructions.
6
Briefly, 1.5 × 10 A549 cells per well were seeded in a six-well plate. Cells were preincubated in
drug-free media at 310 K for 24 h in a 5% CO humidified atmosphere, and then drugs were added
2
at concentrations of 0.25 × IC and 0.5 × IC . After 24 h of drug exposure, cells were washed
50
50
twice with PBS and then incubated with the DCFH-DA probe (10 µM) at 37 °C for 30 min, and
then washed triple immediately with PBS. The fluorescence intensity was analyzed by flow
cytometry (ACEA NovoCyte, Hangzhou, China). Data were processed using NovoExpress™
software. At all times, samples were kept under dark conditions to avoid light-induced ROS
production.
Mitochondrial Membrane Assay. Analysis of the changes of mitochondrial potential in cells
after exposure to metal complex was carried out using the Mitochondrial membrane potential
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201801339
assay kit with JC-1 (Beyotime Institute of Biotechnology, Shanghai, China) according to the
6
manufacturer’s instructions. Briefly, 1.5 × 10 A549 cancer cells were seeded in six-well plates left
to incubate for 24 h in drug-free medium at 310 K in a humidified atmosphere. Drug solutions, at
concentrations of 0.25 × IC50 , 0.5 × IC , 1× IC and 2 x IC50 of complex against A549 cancer
50
50
cells, were added in triplicate, and the cells were left to incubate for a further 24 h under similar
conditions. Supernatants were removed by suction, and each well was washed with PBS before
detaching the cells using trypsin-EDTA. Staining of the samples was done in flow cytometry tubes
protected from light, incubating for 30 min at ambient temperature. The samples were
immediately analyzed by a flow cytometer (ACEA NovoCyte, Hangzhou, China). For positive
controls, the cells were exposed to carbonyl cyanide 3-chlorophenylhydrazone, CCCP (5 μM), for
20 min. Data were processed using NovoExpress™ software.
ASSOCIATED CONTENT
The Supporting Information is available free of charge on the Publications website at DOI: xxx
xxx xxx.
Experimental Section, Fig. S1- Fig. S5. Table S1 – Table S3 (PDF).
AUTHOR INFORMATION
Corresponding author.
Email: liuzheqd@163.com
ORCID
Zhe Liu: 0000-0001-5796-4335
Author Contributions
All authors have given approval to the final version of the manuscript.
Conflicts of interest
There are no conflicts of interest to declare.
ACKNOWLEDGMENTS
We thank the National Natural Science Foundation of China (GrantNo.21671118) and the
Taishan Scholars Program for support.
References
1.
2.
3.
C. A. Puckett, R. J. Ernst and J. K. Barton, Dalton Trans., 2010, 39, 1159-1170.
N. Muhammad and Z. Guo, Curr. Opin. Chem. Biol., 2014, 19, 144-153.
J. X. Liang, H. J. Zhong, G. Yang, K. Vellaisamy, D. L. Ma and C. H. Leung, J. Inorg. Biochem.,
2
017, 177, 276-286.
4.
5.
6.
C.-H. Leung, S. Lin, H.-J. Zhong and D.-L. Ma, Chem. Sci., 2015, 6, 871-884.
T. W. Hambley, Dalton Trans., 2007, 4929-4937.
S. M. Emam, I. E. T. El Sayed and N. Nassar, Spectrochim. Acta. A Mol. Biomol. Spectrosc.,
2
015, 138, 942-953.
M. A. Malik, O. A. Dar, P. Gull, M. Y. Wani and A. A. Hashmi, MedChemComm, 2018, 9,
09-436.
7.
8.
9.
4
Y. Wang, H. Wang, H. Li and H. Sun, A. Mudipalli and J. T. Zelikoff, Springer International
Publishing, Cham, 2017, pp. 199-222.
Q. Cao, Y. Li, E. Freisinger, P. Z. Qin, R. K. O. Sigel and Z. W. Mao, Inorg. Chem. Front., 2017, 4,
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201801339
1
0-32.
R. Guan, Y. Chen, L. Zeng, T. W. Rees, C. Jin, J. Huang, Z. S. Chen, L. Ji and H. Chao, Chem. Sci.,
018, 9, 5183-5190.
10.
2
11.
12.
13.
14.
15.
Y. Huang, W. Huang, L. Chan, B. Zhou and T. Chen, Biomaterials, 2016, 103, 183-196.
M. D. Hall and T. W. Hambley, Coord. Chem. Rev., 2002, 232, 49-67.
R. A. Alderden, M. D. Hall and T. W. Hambley, J. Chem. Educ., 2006, 83, 728.
A. V. Klein and T. W. Hambley, Chem. Rev., 2009, 109, 4911-4920.
R. E. Aird, J. Cummings, A. A. Ritchie, M. Muir, R. E. Morris, H. Chen, P. J. Sadler and D. I.
Jodrell, Br. J. Cancer, 2002, 86, 1652.
1
6.
7.
C. G. Hartinger and P. J. Dyson, Chem. Soc. Rev., 2009, 38, 391-401.
J. Li, L. Guo, Z. Tian, M. Tian, S. Zhang, K. Xu, Y. Qian and Z. Liu, Dalton Trans., 2017, 46,
1
15520-15534.
1
8.
9.
J. Li, M. Tian, Z. Tian, S. Zhang, C. Yan, C. Shao and Z. Liu, Inorg. Chem., 2018, 57, 1705-1716.
M. Tian, J. Li, S. Zhang, L. Guo, X. He, D. Kong, H. Zhang and Z. Liu, Chem. Commun., 2017, 53,
1
12810-12813.
20.
21.
22.
Z. Xu, D. Kong, X. He, L. Guo, X. Ge, X. Liu, H. Zhang, J. Li, Y. Yang and Z. Liu, Inorg. Chem.
Front., 2018, 2100-2105.
L. Zeng, P. Gupta, Y. Chen, E. Wang, L. Ji, H. Chao and Z. S. Chen, Chem. Soc. Rev., 2017, 46,
5771-5804.
L. Biancalana, L. K. Batchelor, T. Funaioli, S. Zacchini, M. Bortoluzzi, G. Pampaloni, P. J. Dyson
and F. Marchetti, Inorg. Chem., 2018, 57, 6669-6685.
2
3.
4.
A.-d. C. Silvia, C. A. L., L.-G. Fernando and S. Luca, Angew. Chem., 2018, 130, 3197-3201.
F. Martínez-Peña, S. Infante-Tadeo, A. Habtemariam and A. M. Pizarro, Inorg. Chem., 2018, 57,
2
5657-5668.
25.
26.
27.
S. Thota, D. A. Rodrigues, D. C. Crans and E. J. Barreiro, J. Med. Chem., 2018, 61, 5805-5821.
G. Sava, S. Pacor, G. Mestroni and E. Alessio, Clin. Exp. Metastasis, 1992, 10, 273-280.
P. Costantini, E. Jacotot, D. Decaudin and G. Kroemer, J. Natl. Cancer Inst., 2000, 92,
1
042-1053.
L. He, Y. Li, C. P. Tan, R. R. Ye, M. H. Chen, J. J. Cao, L. N. Ji and Z. W. Mao, Chem. Sci., 2015, 6,
409-5418.
J. Li, L. Guo, Z. Tian, S. Zhang, Z. Xu, Y. Han, R. Li, Y. Li and Z. Liu, Inorg. Chem., 2018, 57,
3552-13563.
J. Li, Z. Tian, Z. Xu, S. Zhang, Y. Feng, L. Zhang and Z. Liu, Dalton Trans., 2018, 47,
5772-15782.
W. Ma, Z. Tian, S. Zhang, X. He, J. Li, X. Xia, X. Chen and Z. Liu, Inorg. Chem. Front., 2018, 5,
587-2597.
Z. Tian, J. Li, S. Zhang, Z. Xu, Y. Yang, D. Kong, H. Zhang, X. Ge, J. Zhang and Z. Liu, Inorg. Chem.,
018, 57, 10498-10502.
28.
29.
30.
31.
32.
5
1
1
2
2
3
3.
4.
X. Jia, H. Liu, Y. Hu, Q. Dai, J. Bi, C. Bai and X. Zhang, Chinese J. Catal., 2013, 34, 1560-1569.
F. K. Cheung, C. Lin, F. Minissi, A. L. Crivillé, M. A. Graham, D. J. Fox and M. Wills, Org. Lett.,
3
2
007, 9, 4659-4662.
3
5.
6.
F. X. Wang, M. H. Chen, Y. N. Lin, H. Zhang, C. P. Tan, L. N. Ji and Z. W. Mao, ACS Appl. Mater.
Inter., 2017, 9, 42471-42481.
3
J. J. Soldevila-Barreda, I. Romero-Canelón, A. Habtemariam and P. J. Sadler, Nat. Commun.,
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201801339
2
015, 6, 6582-6590.
3
7.
8.
L. Zhe, R. C. Isolda, Q. Bushra, H. J. M., H. Abraha, B. N. P. E., P. A. M., C. G. J. and S. P. J.,
Angew. Chem., 2014, 126, 4022-4027.
3
W. Yang, L. Shenglin, Z. Chi, L. Xingyun, L. Tao, J. Zhongyong, L. Dengqun, T. Xu, L. Lei, W. Yu, C.
Zelin, L. Yunsheng, Y. Fan, G. Yibo and S. Chunmeng, Advanced Materials, 2018, 0, 1800475.
M. T. Gyparaki and A. G. Papavassiliou, Trends Mol. Med., 2014, 20, 239-241.
D. Y. Zhang, Y. Zheng, C. P. Tan, J. H. Sun, W. Zhang, L. N. Ji and Z. W. Mao, ACS Appl. Mater.
Inter., 2017, 9, 6761-6771.
3
9.
0.
4
4
1.
2.
Y. Li, C.-P. Tan, W. Zhang, L. He, L.-N. Ji and Z.-W. Mao, Biomaterials, 2015, 39, 95-104.
J. Ji, X. Li, T. Wu and F. Feng, Chem. Sci., 2018, 9, 5816-5821.
4
This article is protected by copyright. All rights reserved.